Glucagon receptor blockade protects spermatogenesis by enhancing PFKFB3-mediated lactate production in Sertoli cells

Abstract

Background

Male fertility has declined globally. Diabetes and aging are major contributors to the reduced sperm production and testicular dysfunction. Elevated glucagon signaling leads to diabetic complications, yet its impact on male reproductive function remains unclear.

Methods

Streptozotocin-induced type 1 diabetic mice, naturally aged mice, and healthy young male mice were treated with a glucagon receptor (GCGR) monoclonal antibody or control. Sperm quality and testicular outcomes, including histology, oxidative stress, and apoptosis, were assessed. We then generated global Gcgr knockout mice and compared their sperm parameters and testicular outcomes with those of wild-type mice. Mouse spermatogonia cells, spermatocyte cells, Leydig cells, and Sertoli cells were treated with different concentrations of glucagon, and cell proliferation was evaluated. In Sertoli cells, maturation markers and blood-testis barrier (BTB)-associated genes were examined. The histology and function of Sertoli cells were further measured in diabetic or aged mice after GCGR antibody treatment. To investigate the underlying mechanism, we treated Sertoli cells with glucagon, a GCGR antibody, or a GLP-1R antagonist, and measured the lactate content, lactate dehydrogenase activity, and the expression of glucose transporters, glycolysis-associated genes, and lactate dehydrogenase isoforms. We focused on the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). The lactate production and PFKFB3 level were further determined in diabetic or aged mice. Next, by using over-expression or knockdown of Pfkfb3 in glucagon-treated Sertoli cells and constructing Pfkfb3 heterozygous knockout mice, we confirmed the role of PFKFB3 in lactate production and Sertoli-cell function.

Results

In diabetic mice, the GCGR antibody increased sperm concentration (P < 0.001) and reduced testicular oxidative stress and apoptosis. In aged mice, the GCGR antibody improved progressive motility (P = 0.004), with minimal effects on reproductive hormone levels, testicular oxidative stress, and apoptosis. In healthy young mice, the GCGR antibody increased sperm concentration (P = 0.003), progressive motility (P = 0.022), and total motility (P = 0.020). Global Gcgr knockout impaired spermatogenesis despite improved metabolic status, indicating the participation of GCGR signaling in embryonic testicular development and spermatogenesis. Glucagon did not directly alter isolated sperm motility or proliferation of testicular cell lines, but it suppressed Sertoli-cell maturation markers, BTB-associated genes, lactate production, and PFKFB3 expression. These effects were rescued by the GCGR antibody, whereas GLP-1R blockade showed partial or variable protection. Besides, these inhibitory effects of glucagon were rescued by Pfkfb3 overexpression while Pfkfb3 knockdown showed the similar effects with glucagon. In diabetic and aged mice, the GCGR antibody administration restored Sertoli-cell maturation markers and BTB markers, upregulated testicular lactate levels and increased the abundance of PFKFB3. Pfkfb3 heterozygous deletion reduced total motility (P = 0.007) and testicular lactate content (P = 0.013), accompanied by the lower Sertoli-cell markers and BTB-associated genes expression.

Conclusions

Pharmacological GCGR blockade reverses dysfunctional Sertoli cells and restores spermatogenesis in models of diabetes and aging, and PFKFB3-mediated lactate production participated this process. Our study presents a promising therapeutic strategy for male infertility.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12967-026-08588-y.

Introduction

Global fertility rates have significantly declined over the past 20 years, with male factors contributing to 30%–50% of infertility cases [1]. The growing prevalence of diabetes and the trend toward delayed parenthood represent significant risk factors for impaired male fertility [2, 3]. About 35%–51% of men with diabetes are infertile [4], characterized by reduced sperm quality, testicular oxidative stress, and apoptosis [5, 6]. While glycemic control is the cornerstone of diabetes management, the direct impact of most antidiabetic therapies on male reproductive function remains poorly understood [7]. Given the importance of ensuring reproductive health during diabetes management, it is crucial to understand how antidiabetic therapies influence male fertility.

Glucagon, a hormone secreted by pancreatic α-cells, acts via the glucagon receptor (GCGR) to regulate hepatic glucose output [8]. Hyperglucagonemia and heightened GCGR signaling are featured manifestation in diabetes [9]. Consequently, therapeutic strategies targeting GCGR, particularly GCGR monoclonal antibodies (mAbs), have emerged as a promising therapeutic approach to improve glycemic control [10, 11]. Among these antibodies, REMD-477, also known as volagidemab, is a promising one. REMD-477 is a fully human IgG2 anti-GCGR antibody that has been clinically evaluated and shown to reduce insulin requirements and improve glycemic control [12, 13]. Another mAb, REMD 2.59, differs from REMD-477 by only one amino acid (which is not involved in glucagon binding). It also blocks glucagon–GCGR signaling and produces sustained glucose-lowering effects in diabetic rodent models [14, 15], and it has been widely used as a preclinical surrogate for volagidemab [16]. Notably, GCGR inhibition in rodents has been shown to attenuate heart failure progression [17], modulate myocardial energy metabolism [18], and preserve pancreatic β-cell function [15], suggesting that glucagon signaling has broad physiological effects beyond glucose homeostasis. As far as we know, no literature focused on the effect of GCGR signaling on spermatogenesis. Early studies showed Gcgr knockout females were poor breeders, with smaller litters and exhibited persistent hypoglycemia and hyperglucagonemia during pregnancy, accompanied by reduced fetal number, fetal weight, and placental weight [19–21]. Gcgr knockout male mice were only reported to be fertile [19], while no study showed if Gcgr knockout affects spermatogenesis and the fertility outcomes. The potential influence of GCGR signaling and its pharmacological inhibition on adult male reproductive health remains largely unknown.

In this study, we evaluated whether GCGR blockade benefited male reproduction in type 1 diabetic mice and in aged mice, and we constructed global Gcgr knockout mice to define GCGR’s physiological role. We mapped GCGR expression in testes and testicular cell lines, and then we investigated the direct effects of glucagon on sperm motility in isolated mouse sperm and cell proliferation in four testicular cell lines. Next, we focused on the glucagon’s effects on the function of Sertoli cells. We quantified glucagon’s impact on the expression of maturation markers, blood–testis barrier (BTB)–associated genes, and we detected lactate production. Furthermore, we measured lactate dehydrogenase (LDH) activity, and the expression of the glucose transporters, the glycolysis-associated genes and different LDH isoforms, and focused on the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We tested whether GCGR inhibition in diabetic or aged mice could restore Sertoli-cell maturation markers and BTB genes, upregulate testicular lactate, and increase the expression of PFKFB3. Finally, by conducting the overexpression and knockdown experiments in Sertoli cells and constructing the Pfkfb3 heterozygous knockout mice, we confirmed the role of PFKFB3 in lactate production and Sertoli-cell function. Our study establishes a preclinical basis for targeting GCGR in the treatment of infertility, reveals a previously unrecognized role for glucagon in regulating Sertoli cell function, and provides new insights into the crosstalk between metabolic signaling and male reproductive health.

Materials and methods

Animals and interventions

All animal experiments were conducted at Peking University Health Science Center and approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center (Approval number PUIRB-LA2023522). All animals were group-housed conventionally and maintained on a 12-h light/dark cycle at 23 ± 3 °C with free access to food and water.

To generate a type 1 diabetic model, 8-week-old male C57BL/6J mice (purchased from Department of Laboratory Animal Science, Peking University Health Science Center, Beijing, China) were intraperitoneally injected with streptozotocin (125 mg/kg). The diabetic condition was confirmed by monitoring fasting blood glucose levels ≥ 11.1 mmol/L or random blood glucose levels ≥ 16.7 mmol/L at least twice. The mice were divided into two groups based on comparable distributions of body weight and blood glucose levels, with 10 mice in each group. The GCGR mAb group received intraperitoneal injections of GCGR mAb (REMD 2.59, 5 mg/kg, once a week) for 4 weeks. The control group received intraperitoneal injections of IgG (5 mg/kg, once a week). The dose and frequency of GCGR mAb were selected based on previous pharmacological and pharmacokinetic studies of antagonistic GCGR mAb [14, 16, 22].

Fourteen 18-month-old C57BL/6J male mice (Biocytogen Inc., Beijing, China) were used as the naturally aged model. The mice were divided into two groups based on comparable distributions of body weight and blood glucose levels, with 7 mice in each group. Following a one-week adaptation period, the mice were weekly treated with 5 mg/kg GCGR mAb or IgG (as control) for 4 weeks.

Twenty 8-week-old healthy male C57BL/6J mice were used to evaluate the effects of GCGR blockade under physiological conditions. The mice were randomly divided into two groups with comparable body weight and blood glucose levels, with 10 mice in each group. The mice were weekly treated with 5 mg/kg GCGR mAb or IgG (as control) for 4 weeks.

Global Gcgr knockout (Gcgr^−/−) mice were generated by GemPharmatech (Nanjing, China). A CRISPR/Cas9 deletion strategy targeting the mouse Gcgr gene was used (C57BL/6JGpt background): two sgRNAs were designed to excise the genomic segment spanning exon 4 to exon 14 of the Gcgr-201 transcript (ENSMUST00000026119.7), removing ~ 1.3 kb of coding sequence and disrupting receptor function (Fig. S1). Male 12-week-old Gcgr^−/− mice, alongside wild-type littermates as control mice, were used for subsequent experiments (n = 8 per group).

Pfkfb3 heterozygous knockout mice (Pfkfb3^+/−) were obtained from GemPharmatech Co., Ltd. A CRISPR/Cas9 deletion strategy targeting the mouse Pfkfb3 gene was used (C57BL/6JGpt background): two sgRNAs were designed to flank exons 2 and 3 of the Pfkfb3-220 transcript (ENSMUST00000192949.5), removing 223 bp of coding sequence and generating a loss-of-function allele (Fig. S2). Eight-week-old male Pfkfb3^+/− mice (n = 7), alongside wild-type littermates as control mice (n = 5), were used for subsequent experiments.

The reagents used in this experiment are listed in Table S1.

Body weight, blood glucose and glucose tolerance test

The body weight and random blood glucose levels of mice were measured between 8:00 am and 9:00 am. Fasting blood glucose level was measured after a 12-h overnight fasting. Blood glucose level in samples from a tail vein was monitored by OneTouch Ultra glucometer (LifeScan, Milpitas, CA, USA). If the blood glucose level was higher than 33.3 mmol/L (the upper detection limit of the glucometer), 33.3 mmol/L was recorded.

Intraperitoneal glucose tolerance test was conducted in aged mice following a 12-h overnight fasting. Mice were intraperitoneally injected with a 40% (wt/vol) glucose solution at a dose of 2 g/kg, and blood glucose levels were monitored at baseline (before injection), 30, 60, and 120 min post-administration.

Hormone assays

Blood samples were collected into heparinized 1.5 mL microcentrifuge tubes, kept at 4 °C, and centrifuged at 1,000 × g for 15 min within 30 min of collection. Plasma was stored at − 80 °C until analysis. Plasma levels of testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (Elabscience, Wuhan, China) following the manufacturer’s instructions. The intra-assay and inter-assay CVs were 4.44%–5.89% and 5.16%–5.80% for the testosterone assay kit, 4.16%–5.86% and 6.35%–8.22% for the FSH assay kit, and 4.28%–5.68% and 7.85%–8.53% for the LH assay kit, respectively. Absorbance was read at 450 nm on a microplate reader. The kits used in this experiment are listed in Table S1.

Sperm collection and motility assessment

Following the intervention, mice were deeply anesthetized and the cauda epididymides were excised within 1–2 min. Each cauda epididymis was immediately transferred to an Eppendorf tube containing 1 mL pre-warmed (37 °C) human tubal fluid medium (Nanjing Aibei Biotechnology Co. Ltd, Nanjing, China). Samples were labeled only with mouse identification numbers to ensure blinded analysis. The cauda epididymis was gently minced into three pieces and incubated at 37 °C in a 5% CO₂ incubator for 5 min to allow sperm to swim out.

Sperm count and motility were evaluated using a computer-assisted sperm analysis (CASA) system (SSA-II, Gold Edition, SuiJia Software, Beijing, China) according to the WHO laboratory manual (ISO23162:2021) [23]. The CASA system was calibrated according to the manufacturer’s instructions before analysis. All samples were analyzed under controlled temperature conditions using pre-warmed medium and a temperature-controlled chamber maintained at 37 °C. A 10 µL aliquot of sperm suspension was loaded into a 20-µm-depth sperm counting chamber. Six fields were analyzed for each mouse, and at least 200 spermatozoa were counted per sample. The following parameters were evaluated: sperm concentration (10⁶/mL), rapid progressive motility (grade A, %), progressive motility (grade A + B, %), total motility (grade A + B + C, %), velocity along the straight-line path (VSL, µm/s), velocity along the curvilinear path (VCL, µm/s), velocity along the average path (VAP, µm/s), straightness (STR, %), amplitude of lateral head displacement (ALH, µm), and beat-cross frequency (BCF, Hz). The operator performing CASA acquisition and analysis was unaware of the treatment allocation. The reagents used in this experiment are listed in Table S1.

Testicular weight and gonadosomatic index calculation

Immediately after euthanasia, the testes were carefully dissected from each mouse with excess connective tissue removed. The paired testes weight was recorded for each mouse, and the body weight was measured immediately prior to tissue collection. The gonadosomatic index was then calculated as follows:

Gonadosomatic index = (Testes weight (g) / Body weight (g)) × 100%.

Hematoxylin and Eosin (HE) staining

Testes were rapidly dissected under deep anesthesia and fixed in testicular tissue fixation buffer (Servicebio, Wuhan, China) for 24 h. The tissues were dehydrated through an ethanol gradient, embedded in paraffin, and sectioned into 5-µm-thick slices. Paraffin sections were then deparaffinized in xylene and rehydrated through graded ethanol solutions. For morphological assessment, the sections were stained with HE, and images were acquired using a digital slide scanner (NanoZoomer S210, Hamamatsu Photonics K.K., Japan). Histological image analysis was performed by investigators blinded to group allocation. The reagents used in this experiment are listed in Table S1.

Periodic acid-Schiff and hematoxylin (PAS-H) staining

Paraffin-embedded testicular sections were deparaffinized in xylene and rehydrated through graded ethanol solutions. PAS-H staining was performed using a Glycogen Staining Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Briefly, sections were oxidized with periodic acid solution, treated with Schiff reagent, counterstained with hematoxylin, dehydrated, cleared, and mounted. Images were acquired using a digital slide scanner (NanoZoomer S210). PAS-positive acrosomal structures in developing spermatids within seminiferous tubules were evaluated by investigators blinded to group allocation. The reagents used in this experiment are listed in Table S1.

Immunofluorescent and multiplex immunofluorescent staining

For paraffin-embedded tissue sections, sections were deparaffinized, rehydrated, and subjected to antigen retrieval by heating in Tris-EDTA buffer (pH 9.0). For immunofluorescence staining of cultured cells, cell smears were fixed in pre‑cooled methanol for 15 min. Next, sections/cells were permeabilized using 0.2% Triton X-100 in phosphate-buffered saline (pH 7.3), and blocked with 10% goat serum (Zhongshan Biotechnology, Beijing, China) at room temperature for 30 min.

For conventional immunofluorescence staining, sections or cells were incubated with primary antibodies overnight at 4 °C, followed by incubation with the corresponding fluorescent secondary antibodies for 1 h at room temperature. Nuclei were stained using 4’,6-diamidino-2-phenylindole (DAPI; 1 µg/mL; Applygen, Beijing, China).

For multiplex immunofluorescence staining, a tyramide signal amplification-based sequential staining strategy was used because both primary antibodies were raised in rabbit. Briefly, sections were sequentially incubated with rabbit anti-GCGR (Proteintech) or anti-PFKFB3 antibody (Proteintech), and rabbit anti-SOX9 antibody (HuaAn Biotechnology, Hangzhou, China). After incubation with horseradish peroxidase-conjugated secondary antibody, GCGR or PFKFB3, and SOX9 signals were developed using NEON520 tyramide dye (green; Haosaitehua, Beijing, China) and NEON620 tyramide dye (red; Haosaitehua), respectively, according to the manufacturer’s instructions. Heat-mediated antibody stripping was performed between sequential staining cycles to minimize cross-reactivity between rabbit-derived primary antibodies. Nuclei were counterstained with DAPI.

Fluorescent images were captured with either a digital slide scanner (Pannoramic MIDI, 3D HISTECH, Budapest, Hungary) or a laser-scanning confocal microscope (LSM 990, Carl Zeiss Microscopy GmbH, Jena, Germany). Immunofluorescence image analysis was performed by investigators blinded to group allocation. The reagents used in this experiment are listed in Table S1. The antibodies used in this experiment are listed in Table S2.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay for apoptosis detection

Paraffin-embedded testicular sections were deparaffinized, rehydrated, and permeabilized using proteinase K (Beyotime, Shanghai, China) at room temperature for 30 min. Apoptotic cells were detected using a One Step TUNEL Apoptosis Assay Kit (Beyotime) following the manufacturer’s instructions. Then sections were counterstained with DAPI for nuclear visualization. Fluorescent images were obtained using the digital slide scanner (Pannoramic MIDI, 3DHISTECH). Cells exhibiting green fluorescence were classified as TUNEL-positive. The proportion of TUNEL-positive seminiferous tubules was calculated as (number of TUNEL-positive tubules / total number of tubules) × 100%. The proportion of TUNEL-positive cells was calculated as (number of TUNEL-positive cells / total cell count) × 100%. Immunofluorescence image analysis was performed by investigators blinded to group allocation. The kits used in this experiment are listed in Table S1.

Oxidative stress assessments

Testicular tissue samples were homogenized in ice-cold saline. Oxidative stress markers were quantified using commercial assay kits according to manufacturers’ protocols. Total antioxidant capacity was expressed as Trolox equivalents normalized to total protein concentration, based on the 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization method (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). Superoxide dismutase (SOD) activity was measured using the water-soluble tetrazolium salt method, expressed as U/mg protein (Nanjing Jiancheng Bioengineering Institute). Malondialdehyde (MDA) content was determined via the thiobarbituric acid assay, expressed as nmol/mg protein (Nanjing Jiancheng Bioengineering Institute). All values of total antioxidant capacity, SOD activity, and MDA content were normalized to total protein concentration. The SOD/MDA ratio was calculated using the protein-normalized SOD activity and MDA content. The intra-assay and inter-assay CVs were 3.6% and 6.4% for the total antioxidant capacity assay kit, 5.50% and 3.32% for the SOD activity assay kit, and 2.3% and 5.34% for the MDA assay kit, respectively. The kits used in this experiment are listed in Table S1.

In vitro sperm treatment

Spermatozoa were collected as described and incubated at 37 °C in a humidified 5% CO₂ incubator. Spermatozoa were treated with glucagon (100 nmol/L; R&D Systems, Minneapolis, MN, USA) or phosphate-buffered saline (as vehicle control) for 30 min. The glucagon dose (100 nmol/L) was selected based on previous studies [11, 24]. Immediately after incubation, sperm motility parameters were measured using a CASA system as described above. The reagents used in this experiment are listed in Table S1.

Cell culture and intervention

Mouse spermatogonia (GC-1), spermatocyte (GC-2), Leydig (TM3), and Sertoli (TM4) cell lines were generously provided by Prof. Hui Jiang from the Peking University First Hospital (Beijing, China) (Table S3). GC-1 and GC-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin (Gibco). Cells were passaged at a 1:6 ratio upon reaching 80–90% confluence. TM3 and TM4 cells were cultured in commercially prepared complete DMEM-based medium containing 5% horse serum, 2.5% fetal bovine serum, and 1% penicillin-streptomycin (Procell, Wuhan, China) and passaged at a 1:4 dilution. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

Leydig (TM3) and Sertoli (TM4) cells were treated with different concentrations of glucagon (0, 0.1, 1, 10, 100, 500 nmol/L) with or without a glucagon-like peptide-1 receptor (GLP-1R) antagonist exendin 9–39 (Bachem, Bubendorf, Switzerland; 200 nmol/L) for 72 h, and cell proliferation was detected to assess the possible GLP-1R involvement.

TM4 cells were treated with vehicle control, glucagon (100 nmol/L), glucagon + GCGR mAb (REMD 2.59; 1,000 nmol/L), or glucagon + Exendin 9–39 (200 nmol/L) for 24 h, and the Sertoli-cell’s function was detected to determine the contributing effects of GCGR and GLP-1R. GCGR mAb or Exendin 9–39 was added 2 h before glucagon. The glucagon concentration of 100 nmol/L was selected as a pharmacological GCGR-activation challenge, rather than to mimic physiological circulating glucagon levels, to ensure sufficient receptor engagement and detectable downstream signaling in cultured Sertoli cells, particularly considering potential differences in receptor abundance, peptide stability, and cell-type-specific responsiveness under in vitro conditions. Similar glucagon concentrations have been commonly used in cell-based GCGR signaling studies to evaluate downstream cyclic adenosine monophosphate (cAMP) production, metabolic responses, and transcriptional regulation [11, 25–27].

To further investigate the effects of PFKFB3, TM4 cells were transfected with either a Pfkfb3-overexpressing plasmid or an empty vector control, or with Pfkfb3-specific siRNA or a non-targeting scramble siRNA. After transfection for 24 h, the cells were treated with glucagon (100 nmol/L) or vehicle for an additional 24 h. Subsequently, both cells and culture supernatants were collected for further analysis. The reagents used in this experiment are listed in Table S1. The sequences of si-Pfkfb3 and scramble siRNA control are listed in Table S4.

Cell proliferation assays

GC-1 and GC-2 cells (2 × 10³ cells/well) were seeded in 96-well plates and treated for 24, 48, or 72 h. Cell viability was assessed using Cell counting kit-8 (CCK-8, Dojindo, Japan), with absorbance measured at 450 nm. TM3 and TM4 cells (5 × 10³ cells/well) were cultured for 72 h, and the proliferation were monitored by using the IncuCyte S3 live-cell analysis system (Sartorius, Göttingen, Germany). Cell confluence was quantified by normalization to initial confluence at 0 h, with images captured every 12 h.

RNA extraction, reverse transcription, and real-time quantitative PCR (qPCR)

Total RNA was extracted using Trizol (Thermo Fisher Scientific, Waltham, MA, USA) and reverse-transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time qPCR was performed using Thunderbird SYBR qPCR Mix (TOYOBO, Osaka, Japan) on a QuantStudio5 system (Thermo Fisher Scientific). Primers were synthesized by Tsingke Biotech (Beijing, China), and the gene expression was normalized to Actb and quantified using the 2(^−ΔΔCt) method. The reagents and kits used in this experiment are listed in Table S1. The primers used in PCR are listed in Table S5.

Protein extraction and western blot analysis

Testicular tissues or cells were homogenized in ice-cold radioimmunoprecipitation assay buffer (Applygen) containing protease inhibitors and phosphatase inhibitors (Applygen). After centrifugation (12,000 × g, 4 °C, 15 min), protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Proteins (40 µg per lane) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (MilliporeSigma). Membranes were blocked with 5% bovine serum albumin for 1 h at room temperature, incubated overnight at 4 °C with primary antibodies, followed by incubation with horseradish peroxidase–conjugated secondary antibodies (1 h, room temperature). Protein signals were detected using chemiluminescence (MilliporeSigma) and quantified using Image J. The target proteins were normalized to β-actin. For western blot analyses, all quantified samples represented biological replicates. The reagents and kits used in this experiment are listed in Table S1. The antibodies used in this experiment are listed in Table S2.

Measurement of lactate content and LDH activity

Cell culture supernatants and testicular homogenates were centrifuged (1,000 × g, 10 min), and lactate concentration was measured using a lactate assay kit (Nanjing Jiancheng Bioengineering Institute) at 530 nm. LDH activity was assessed using an LDH assay kit (Nanjing Jiancheng Bioengineering Institute) at 450 nm. Results were normalized to total protein concentration. The intra-assay and inter-assay CVs were 5.74% and 3.1% for the lactic acid assay kit, 4.95% and 2.7% for the LDH assay kit, respectively. The kits used in this experiment are listed in Table S1.

Intracellular cAMP assay

To inhibit cAMP degradation, 3-isobutyl-1-methylxanthine (IBMX; 500 µmol/L; MilliporeSigma) was added 15 min before treatment and maintained during the subsequent period. Cells were stimulated for 15 min, and then were washed three times with cold phosphate-buffered saline and lysed. The lysates were subjected to two freeze–thaw cycles and centrifuged at 600 × g for 10 min at 4 °C. The resulting supernatants were collected for cAMP measurement using a competitive enzyme immunoassay kit (R&D Systems) according to the manufacturer’s instructions. Absorbance was measured at 450 nm with wavelength correction at 540 nm. cAMP concentrations were calculated from a four-parameter logistic standard curve and normalized to the corresponding cell counts. The reagents and kit used in this experiment are listed in Table S1.

Statistical analysis

Data normality was assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated using Levene’s test or Brown–Forsythe test. Normally distributed data are presented as mean ± standard deviation (SD). For comparisons between two groups, normally distributed data with equal variances were analyzed using two-tailed unpaired Student’s t-tests, while normally distributed data with unequal variances were analyzed using Welch’s t-test. For comparisons among multiple groups, normally distributed data with equal variances were analyzed using one-way or two-way ANOVA followed by appropriate post hoc tests, including Tukey’s, Dunnett’s, or Fisher’s LSD tests. For normally distributed data with unequal variances, Welch’s ANOVA followed by Dunnett’s T3 or Tamhane’s T2 post hoc test was used. All statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was defined as P < 0.05.

Results

GCGR mAb restores glycemic control and improves sperm quality in type 1 diabetic mice

First, we investigated the effects of GCGR blockade in type 1 diabetic mice. We found that four-week treatment with GCGR mAb increased body weight, testicular weight and reduced random and fasting blood glucose levels compared with the control group (Fig. 1A–D).

Fig. 1.

GCGR blockade restores glycemic control and improves sperm quality in type 1 diabetic mice. Eight-week-old male C57BL/6J mice were injected with streptozotocin to induce a type 1 diabetic model. The diabetic mice were divided into two groups and were weekly treated with a GCGR monoclonal antibody (mAb, 5 mg/kg), or the IgG control (Ctrl, 5 mg/kg) for 4 weeks (n = 10 per group).A: Body weight. B: Random blood glucose. The arrowhead indicates the glucometer's upper detection limit of 33.3 mmol/L. C: Fasting blood glucose. D: Testicular weight. E–N: Sperm concentration and motility were evaluated using a computer-assisted sperm analysis system. E: Sperm concentration. F: Rapid progressive motility (grade A sperm). G: Progressive motility (grade A + B sperm). H: Total motility (grade A+B+C sperm). I: Velocity along the straight‑line path (VSL). J: Velocity along the curvilinear path (VCL). K: Velocity along the average path (VAP). L: Straightness (STR). M: Amplitude of lateral head displacement (ALH). N: Beat-cross frequency (BCF). Data are expressed as the mean ± SD. Statistical analyses included two-way ANOVA followed by Tukey’s post hoc test, unpaired two-tailed Student’s t-test or Welch’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control group

Subsequently, we evaluated the sperm quality using CASA system. Compared with mice in the control group, the mice in GCGR mAb group demonstrated significantly higher sperm concentration (37.85 ± 7.54 million/mL vs. 20.68 ± 5.41 million/mL, P < 0.001), rapid progressive motility (12.84 ± 4.11% vs. 1.62 ± 0.67%, P < 0.001), progressive motility (45.57 ± 7.33% vs. 28.69 ± 5.05%, P < 0.001), and total motility (48.03 ± 8.00% vs. 39.95 ± 5.84%, P = 0.0188) (Fig. 1E–H), indicating improved sperm activity and forward movement efficiency. Sperm velocity parameters—VSL (22.44 ± 4.70 μm/s vs. 13.15 ± 4.41 μm/s, P < 0.001), VCL, and VAP—were also markedly enhanced by GCGR mAb (Fig. 1I–K), highlighting greater sperm vigor. Additionally, significant improvements of STR (indicating more linear movement), and ALH (reflecting increased sperm motility vigor) were observed (Fig. 1L, M), while no significant difference of BCF (reflecting sperm flagellar beating intensity and motility potential) was shown in mAb group when compared to the control group (Fig. 1N).

To determine whether the improvement in sperm quality was associated with systemic endocrine changes, we measured circulating reproductive hormone levels in diabetic mice and aged mice. GCGR mAb treatment did not significantly alter plasma testosterone, FSH, or LH levels compared with control group (Fig. S3 A–C).

GCGR mAb improves seminiferous tubule integrity and reduces oxidative stress and apoptosis in type 1 diabetic mice

Next, we evaluated the effects of GCGR blockade on testicular morphology. Histological analysis demonstrated severe structural disruption in the seminiferous tubules of type 1 diabetic mice, characterized by fissures, decreased epithelial thickness, disorganized cellular arrangements, and substantial loss of spermatogenic cells and spermatids. Numerous intercellular vacuoles and cell debris were visible within the tubular lumens. Treatment with GCGR mAb significantly improved structural integrity, restored organized cellular arrangement, and prevented cellular loss in seminiferous tubules (Fig. 2A). To further characterize spermatogenic status, PAS-H staining was performed to visualize acrosomal development in seminiferous tubules. In the control mice, PAS-positive acrosomal structures were relatively sparse and irregular, accompanied by disorganized spermatogenic epithelium. GCGR mAb-treated diabetic mice showed more organized germ-cell layers and more evident PAS-positive acrosomal structures in developing spermatids within the seminiferous tubules (Fig. 2B). These observations further support that GCGR mAb treatment ameliorates diabetes-associated impairment of spermatogenesis.

Fig. 2.

GCGR blockade improves seminiferous tubule integrity and acrosomal structures and reduces apoptosis and oxidative stress in type 1 diabetic mice. Eight-week-old male C57BL/6J mice were used to establish a type 1 diabetic model and then treated with a GCGR monoclonal antibody (mAb) or the IgG control (Ctrl). Testes were collected for histology (A, B), apoptosis analyses (C–I), and oxidative-stress assays (J–M). A: Representative hematoxylin and eosin (HE)-stained testicular sections. Dashed-boxed regions are shown at higher magnification below. Scale bar = 50 μm. B: Representative periodic acid–Schiff–hematoxylin (PAS-H)-stained testicular sections. Dashed-boxed regions are shown at progressively higher magnifications from top to bottom. Arrows indicate PAS-positive acrosomal structures in developing spermatids. Scale bar = 50 μm. C: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Scale bar = 50 μm. D: Percentage of TUNEL-positive seminiferous tubules [(number of TUNEL-positive tubules / total number of tubules) × 100%]. E: Percentage of TUNEL-positive cells [(number of TUNEL-positive cells / total cell count) × 100%]. n = 5. F–I: The protein levels of apoptosis regulator BCL2 (an anti-apoptosis protein) and BAX (a pro-apoptosis protein) were measured by western blot and quantified using Image J software. n = 3–4. J: Trolox-equivalent antioxidant capacity (a commonly used Trolox-based assay for total antioxidant capacity). K: Superoxide dismutase (SOD) activity. L: Malondialdehyde (MDA) content. M: SOD/MDA ratio. n= 5. Data are expressed as the mean ± SD. Statistical analyses were performed by two-tailed unpaired Student’s t-test or Welch’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control group

Then the apoptosis markers were assessed. The control diabetic mice exhibited numerous TUNEL-positive germ cells along the basement membrane of seminiferous tubules, indicative of elevated apoptosis (Fig. 2C). GCGR mAb treatment markedly reduced percentage of TUNEL-positive seminiferous tubules (7.56 ± 1.75% vs. 15.89 ± 4.26%, P = 0.009) and TUNEL-positive cells (4.83 ± 0.81% vs. 9.88 ± 2.40%, P = 0.007) (Fig. 2D, E). Additionally, GCGR mAb significantly increased anti-apoptotic B-cell lymphoma 2 (BCL2) expression and decreased pro-apoptotic Bcl-2–associated X protein (BAX) expression, resulting in a higher BCL2/BAX ratio (2.49 ± 0.35 vs. 1.26 ± 0.32, P = 0.0049) and reduced apoptotic signaling (Fig. 2F–I).

Oxidative stress was also evaluated as a potential mechanism of GCGR mAb treatment. Compared with the control group, GCGR mAb treatment significantly increased total antioxidant capacity (134.40 ± 10.44 mmol/mg protein vs. 97.79 ± 13.89 mmol/mg protein, P = 0.0015) and SOD activity (126.90 ± 4.61 U/mg protein vs. 100.90 ± 5.55 U/mg protein, P < 0.001), indicating enhanced antioxidant defenses (Fig. 2J, K). GCGR mAb administration reduced MDA levels (0.62 ± 0.090 nmol/mg protein vs. 1.02 ± 0.23 nmol/mg protein, P = 0.014), reflecting decreased lipid peroxidation (Fig. 2L). An elevated SOD/MDA ratio further confirmed the improved oxidative stress balance in the GCGR mAb group, compared with that in the control group (207.50 ± 26.54 U/nmol vs. 101.50 ± 15.88 U/nmol, P < 0.001; Fig. 2M).

GCGR mAb improves glycemic control and sperm quality in aged mice

The effects of GCGR blockade were further investigated in aged mice. Results showed that GCGR mAb treatment did not significantly alter body weight but induced a sustained reduction in blood glucose levels without hypoglycemia compared with the control treatment (Fig. 3A, B). GCGR mAb group displayed the significantly lower glucose concentrations at all time points in glucose tolerance test and a reduced area under the curve compared to controls (Fig. 3C, D).

Fig. 3.

GCGR blockade improves glycemic control and sperm quality in aged mice. Eighteen-month-old male C57BL/6J mice were divided into two groups and treated with a GCGR monoclonal antibody (mAb, 5 mg/kg) or IgG control (Ctrl, 5 mg/kg) for 4 weeks (n = 7 per group). A: Body weight. B: Random blood glucose. C: Glucose level during intraperitoneal glucose tolerance test (IPGTT) with 2 g/kg glucose load. D: Area under the curve (AUC) during the IPGTT. E–N: Sperm concentration and motility were evaluated using a computer-assisted sperm analysis system. E: Sperm concentration. F: Rapid progressive motility (grade A sperm). G: Progressive motility (grade A + B sperm). H: Total motility (grade A+B+C sperm). I: Velocity along the straight‑line path (VSL). J: Velocity along the curvilinear path (VCL). K: Velocity along the average path (VAP). L: Straightness (STR). M: Amplitude of lateral head displacement (ALH). N: Beat-cross frequency (BCF). Data are expressed as the mean ± SD. Statistical analyses were performed by two-tailed unpaired Student’s t-test. **P < 0.01, and ***P < 0.001 vs. the control group

While GCGR mAb has no effect on sperm concentration, it markedly enhanced multiple sperm motility parameters, including progressive motility (33.88 ± 7.24% vs. 20.29 ± 7.06%, P = 0.0039) and total motility (37.64 ± 7.49% vs. 22.76 ± 7.39%, P = 0.0028) (Fig. 3E–H). Detailed velocity analysis demonstrated significant increases in VSL (14.35 ± 3.19 μm/s vs. 8.55 ± 2.77 μm/s, P = 0.0035), VCL, VAP, and STR (Fig. 3I–L). Moreover, indicators of sperm vigor, specifically ALH and BCF, were significantly elevated in GCGR mAb-treated mice (Fig. 3M, N). These improvements in sperm quality mirrored those observed in type 1 diabetic mice (Fig. 1E–N). Similarly, GCGR mAb treatment did not significantly alter circulating testosterone, FSH, or LH levels in aged mice (Fig. S3D–F).

Taken together, these results indicate that GCGR mAb exerts reproductive benefits extending beyond lowering blood glucose or systemic hormone changes, highlighting its therapeutic potential to improve reproductive health, even when metabolic dysfunction is not the primary contributing factor.

GCGR mAb improves late spermatogenic features while has minimal effects on oxidative stress and apoptosis in aged mice

Following, we evaluated the testicular morphology. In aged mice, the testes displayed intact seminiferous tubule architecture, with no apparent abnormality in cellular arrangement, thickness, or morphology, and there was no significant difference between the GCGR mAb-treated group and the control group (Fig. 4A). PAS-H staining exhibited a more organized seminiferous epithelium and more prominent PAS-positive acrosomal structures in developing spermatids in the GCGR mAb-treated group, compared with those in the control group (Fig. 4B). These findings suggest that GCGR mAb treatment may improve late spermatogenic features, particularly spermatid maturation and acrosome formation.

Fig. 4.

GCGR blockade improves spermatid maturation and acrosome formation and has minimal effects on apoptosis and oxidative stress in aged mice. Eighteen-month-old male C57BL/6J mice were treated with a GCGR monoclonal antibody (mAb) or IgG control (Ctrl). After a 4-week treatment, testes were harvested for histology (A, B), apoptosis analyses (C–E), and oxidative-stress assays (F–I). A: Representative hematoxylin and eosin (HE)-stained testicular sections. Dashed-boxed regions are shown at higher magnification below. Scale bar = 50 μm. B: Representative periodic acid–Schiff–hematoxylin (PAS-H)-stained testicular sections. Dashed-boxed regions are shown at progressively higher magnifications from top to bottom. Arrows indicate PAS-positive acrosomal structures in developing spermatids. Scale bar = 50 μm. C–E: Protein levels of apoptosis regulator BCL2 and BAX were measured by western blot and quantified using Image J software. n = 4. F: Trolox-equivalent antioxidant capacity (a commonly used Trolox-based assay for total antioxidant capacity). G: Superoxide dismutase (SOD) activity. H: Malondialdehyde (MDA) content. I: SOD/MDA ratio. n = 7. Data are expressed as the mean ± SD. Statistical analyses were performed by two-tailed unpaired Student’s t-test

Apoptotic signaling analysis showed no significant changes in BCL2 (1.09 ± 0.26 vs. 1.32 ± 0.19, P = 0.21) or BAX expression (0.81 ± 0.12 vs. 0.82 ± 0.20, P = 0.98) levels between the GCGR mAb-treated and the control group (Fig. 4C–E). Meanwhile, markers indicative of oxidative stress, including total antioxidant capacity (123.30 ± 11.37 U/mg protein vs. 124.90 ± 13.77 U/mg protein , P = 0.82), SOD activity (103.99 ± 19.80 U/mg protein vs. 119.27 ± 11.54 U/mg protein, P = 0.10), MDA content (0.68 ± 0.098 mmol/mg protein vs. 0.67 ± 0.14 mmol/mg protein, P = 0.83) and SOD/MDA ratio (Fig. 4F–I), showed no significant difference between GCGR mAb treatment and the control treatment. These findings suggest that GCGR mAb treatment does not substantially influence oxidative stress and apoptosis in aged mice, likely due to their relatively preserved metabolic function and testicular structure during aging.

Collectively, these data imply that GCGR mAb-mediated improvements in sperm quality and spermatogenic features in aged mice occur independently of alterations in testicular oxidative stress or apoptosis, highlighting alternative mechanisms underlying its reproductive benefits in the absence of pronounced metabolic dysfunction.

GCGR mAb treatment improves sperm quality in healthy young mice

To further determine whether GCGR blockade affects male reproductive parameters under physiological conditions, healthy 8-week-old male C57BL/6J mice were treated with GCGR mAb or IgG control for 4 weeks. GCGR mAb treatment did not significantly alter body weight during the intervention period (Fig. S4A), but moderately reduced both random and fasting blood glucose levels without inducing hypoglycemia (Fig. S4B, C).

GCGR mAb treatment increased testes weight and the gonadosomatic index compared with control treatment (Fig. S4D, E). GCGR mAb significantly increased sperm concentration (38.15 ± 12.77 million/mL vs. 22.64 ± 6.24 million/mL , P = 0.003; Fig. S4F). Progressive motility was also significantly enhanced in GCGR mAb-treated mice (28.69 ± 9.54% vs. 19.05 ± 7.51%, P = 0.022; Fig. S4H), accompanied by increased total motility (38.87 ± 9.12% vs. 28.98 ± 8.19%, P = 0.020; Fig. S4I). Sperm velocity parameters were similarly improved, including VSL, VCL and VAP (Fig. S4J–L). In addition, GCGR mAb increased STR, ALH and BCF (Fig. S4M–O). These findings indicate that short-term pharmacological GCGR blockade improves sperm concentration and multiple motility-related parameters in healthy young adult mice.

Global Gcgr knockout reduces sperm concentration and motility without gross testicular changes

To further explore the role of GCGR signaling in male reproductive health, we constructed global Gcgr^−/− mice. Consistent with previous reports, Gcgr^−/− mice exhibited significantly reduced random and fasting blood glucose levels compared to wild-type littermates (Fig. S5A, B), confirming the established metabolic phenotype following GCGR deletion [19]. The body weight and gonadosomatic index did not differ significantly between Gcgr^−/− and wild-type mice (Fig. S5C–E). Despite these metabolic advantages, Gcgr^−/− mice showed impairments in sperm quality. Sperm concentration (17.94 ± 8.18 million/mL vs. 34.94 ± 12.26 million/mL, P = 0.0057) in the Gcgr^−/− mice was lower than that in the wild-type mice (Fig. S5F). Several indicators of sperm motility [including the total motility (33.50 ± 4.11% vs. 39.08 ± 5.39%, P = 0.035)] and indicators of directional and vigorous sperm movement (including STR and BCF) were reduced; while several indicators of sperm motility (including rapid progressive motility, progressive motility) and sperm movement (including VSL, VCL, VAP, ALH) remained unchanged in the Gcgr^−/− mice compared the wild-type mice (Fig. S5G–O). Besides, we compared the livebirth from wild-type male × wild-type female, heterozygous Gcgr knockout (Gcgr^+/−) male × wild-type female, Gcgr^+/− male × Gcgr^+/− female, Gcgr^−/− male× Gcgr^+/− female. We found there was no difference among these mice. These results suggested that although the sperm concentration and several parameters of sperm motility are decreased in Gcgr knockout mice, they did not result in a reduced fertile outcome (Fig. S6).

Histological examination of testicular tissue demonstrated the intact seminiferous tubule architecture and no obvious abnormalities in Gcgr^−/− mice (Fig. S5P), indicating that impaired sperm quality after GCGR knockout from the beginning of life was not attributable to gross structural abnormalities.

Direct glucagon intervention has limited impact on sperm motility but may modulate Sertoli cell function

To define the direct role of glucagon signaling in male reproduction, we first assessed GCGR expression in testes and different testicular cells. We found GCGR was broadly expressed in human and mouse testes, and it could be detected in mouse testis, Sertoli cells (TM4), Leydig (TM3) and germ cell lines (GC-1, GC-2) (Fig. S7A–D, Fig. 5A). We subsequently tested glucagon’s direct impact and found that glucagon treatment did not alter isolated mouse sperm motility (Fig. 5B–J) and even a wide range of concentrations of glucagon did not affect the cell proliferation in any testicular cell line (Fig. 5K–N), suggesting that glucagon has limited direct effects on these processes. Glucagon has been identified as a GCGR and GLP-1R dual agonist [28, 29], and considering the established protective roles of GLP-1R signaling [30], we examined the possible GLP-1R involvement. We found GLP-1R expression was abundant in Leydig and Sertoli cells (Fig. S8A). Therefore, to investigate whether GLP-1R signaling mediates the effects of glucagon, we treated TM3 and TM4 cells with glucagon combined with the GLP-1R antagonist exendin 9–39 and found no influence on cell proliferation (Fig. S8B–C). These results suggested that glucagon might affect other biological process rather than the cell proliferation.

Fig. 5.

Direct glucagon intervention has limited impact on sperm motility but modulates Sertoli cell function. A: The expression levels of glucagon receptor (GCGR) were assessed in mouse testicular tissues and cell lines by western blot. Mouse liver tissue and human liver cell line HepG2 were used as positive controls for GCGR expression. B–J: Sperm were isolated from 12-week-old male C57BL/6J mice and treated in vitro with glucagon or phosphate-buffered saline (as vehicle control, Ctrl). The motility was then assessed by a computer-assisted sperm analysis system (n = 8 per group). K–N: Mouse spermatogonia cell line GC-1 cells (GC-1), spermatocyte cell line GC-2 cells (GC-2), Leydig cells (TM3) and Sertoli cells (TM4) were treated with different concentrations of glucagon (GCG). The cell proliferation was evaluated using the CCK8 assay (K–L) or continuous live cell analysis system-the IncuCyte S3 (M–N). n = 3 – 4. O–T: TM4 cells were treated with different concentrations of glucagon for 48 h. The mRNA levels of the Sertoli cell specific markers (O–P) and the blood–testis barrier-associated genes (Q–T) were analyzed by reverse transcription qPCR. n = 4. Data are presented as mean ± SD. Statistical analyses included two-tailed unpaired Student’s t-test; two-way ANOVA followed by Tukey’s post hoc tests; and one-way ANOVA followed by Tukey’s post hoc test or Welch’s ANOVA followed by Tamhane's T2 post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control group

Because Sertoli cells are essential for maintaining the seminiferous tubule microenvironment and overall reproductive function, we next focused on Sertoli cells. Using immunofluorescence, we found GCGR was co-localized with the Sertoli-cell marker SOX9 in human testes, diabetic mouse testes, aged mouse testes, and in TM4 cells (Fig. S7A–D). In addition, GCGR mAb treatment increased testicular GCGR protein abundance in both diabetic and aged mice, while SOX9 protein expression was significantly increased in diabetic mice (Fig. S7E, F). Then we assessed Sertoli-cell functions—the maturation markers and BTB integrity—to test the direct glucagon’s effects. Glucagon treatment significantly downregulated the Sertoli cell marker Gata4 as well as the BTB-associated genes gap junction protein alpha 1 (Gja1, encoding Connexin 43) and claudin 11 (Cldn11, encoding Claudin 11) in Sertoli cells; while glucagon did not significantly affect the expression of another Sertoli cell marker Wt1, nor did it alter the expression of BTB-associated junctional components tight junction protein 1 (Tjp1, encoding ZO‑1) and cadherin 2 (Cdh2, encoding N‑cadherin) (Fig. 5O-T). Collectively, these data indicate that although glucagon exerts minimal direct effects on sperm motility and testicular cell proliferation, it can selectively modulate Sertoli‑cell maturation and BTB–related gene expression, suggesting a subtle but specific role in testicular homeostasis.

GCGR mAb increases expression of Sertoli-cell markers and BTB–associated genes in type 1 diabetic mice and aged mice

To assess the impact of glucagon signaling on the BTB in diabetic or aged mice, we evaluated Sertoli-cell markers and BTB components in testes from GCGR mAb or control mice. GCGR mAb increased the expression of Sertoli-cell maturation markers (Wt1 and Gata4) and BTB–associated genes (Gja1 and Tjp1) in diabetic mice (Fig. 6A–F). Immunofluorescence staining and western blot analysis further confirmed the enhanced protein expression of ZO1 (encoded by Tjp1) and Connexin 43 (encoded by Gja1) within seminiferous tubules (Fig. 6G, H), suggestive of the strengthened barrier structure. Similarly, GCGR mAb increased the expression of Gata4, ZO1 and Connexin 43 in aged mice (Fig. 6I–P). Together, these data indicated that GCGR mAb increased markers of Sertoli-cell maturation and the BTB in both models, underscoring GCGR mAb’s therapeutic potential in addressing various reproductive health conditions.

Fig. 6.

GCGR blockade increases the expression of Sertoli-cell specific markers and BTB–associated genes in testes of type 1 diabetic mice and aged mice. Streptozotocin-induced diabetic mice (A–H) and 18-month-old male C57BL/6J mice (I–P) received treatment with a GCGR monoclonal antibody (mAb), or IgG control (Ctrl). After a 4-week treatment, testes were collected. A–F, I–N: The mRNA levels of Sertoli-cell specific markers [Wt1 (A, I), Gata4 (B, J)] and blood–testis barrier (BTB) components [Gja1 (C, K), Cldn11 (D, L), Tjp1 (E, M), and Cdh2 (F, N)] were detected by reverse transcription qPCR. n = 4–5. G, O: Representative images showing the immunofluorescence staining of BTB–associated protein ZO1 and Connexin 43 in testicular tissues. Scale bar = 50 μm. H, P: The protein levels of ZO1 and Connexin 43 were detected by western blot and quantified using Image J software. n = 3. Data were presented as mean ± SD. Statistical analyses were performed using two-tailed unpaired Student’s t-test or Welch’s t-test. *P < 0.05, **P < 0.01 vs. the control group

Glucagon suppresses glycolytic flux and lactate production in Sertoli cells

Given that lactate supplied by Sertoli cells is essential for sustaining normal spermatogenesis and the maintenance of germ cell function [31], we next assessed the effect of glucagon on the lactate production of Sertoli cells. TM4 Sertoli cells showed a significant dose- and time-dependent reduction in lactate production, whereas LDH activity remained unaffected (Fig. 7A, B).

Fig. 7.

Glucagon suppresses glycolytic flux and lactate production in Sertoli cells. Mouse Sertoli cell line TM4 cells were treated with different concentrations of glucagon (GCG) for 24, 48 or 72 h. A–B: Lactate content (A) and LDH activity (B) were measured using commercial colorimetric assay kits. C–K: The mRNA levels of the transporters (C–E), the glycolysis-associated genes (F–H) and different LDH isoforms (I–K) were analyzed by reverse transcription qPCR. L–N: The protein levels of PFKFB3 and LDHA were assessed by western blot and quantified using Image J software. n = 4–5. Data were presented as mean ± SD. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test or Welch’s ANOVA followed by Dunnett’s T3 or Tamhane's T2 post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control group

Sertoli cells metabolize glucose predominantly into lactate, which serves as a major energy substrate for the developing germ cells, through a process involving glucose transporter 1/glucose transporter 3‑mediated glucose uptake, glycolysis, LDH–catalyzed conversion, and subsequent export via monocarboxylate transporter 4 [32, 33]. Next, we profiled the expression of transporters, key glycolytic genes and lactate metabolism-related genes. Glucagon did not change the expression of transporters, including Slc2a1 (encoding glucose transporter 1), Slc2a3 (encoding glucose transporter 3), and Slc16a3 (encoding monocarboxylate transporter 4) (Fig. 7C–E). Although glucagon had no effects on the mRNA levels of glycolytic enzymes genes, including hexokinase 2 (Hk2), pyruvate kinase isozyme M2 (Pkm2), and LDH isoforms (Ldha, Ldhb, Ldhc), it downregulated the expression of Pfkfb3, a critical regulatory enzyme controlling glycolytic flux, in a dose-dependent manner (Fig. 7F-K). The protein level of PFKFB3 was also downregulated by glucagon while LDHA protein expression remained unchanged (Fig. 7L-N).

We next assessed the role of GCGR and GLP-1R on Sertoli-cell metabolic and functional readouts. In TM4 cells, glucagon increased intracellular cAMP levels, reduced lactate production, and upregluated Pfkfb3 expression. GCGR mAb reversed these glucagon-induced changes. The GLP-1R antagonist also attenuated glucagon-induced cAMP accumulation and reversed the reduction in lactate production and Pfkfb3 expression(Fig. S9A–C). In addition, glucagon downregulated several Sertoli-cell markers and BTB-associated genes, including Wt1, Gata4, Gja1 and Tjp1. GCGR mAb clearly rescued these changes, whereas the GLP-1R antagonist showed partial or variable effects (Fig. S9D–I). These results suggest that GLP-1R signaling may contribute to selected glucagon-induced responses in Sertoli cells, but does not fully account for the Sertoli-cell dysfunction induced by glucagon. GCGR blockage might be a more suitable strategy for male reproductive protection.

Together, these findings suggest that glucagon significantly inhibits glycolytic flux and lactate production in Sertoli cells primarily through GCGR and PFKFB3.

GCGR mAb increases testicular lactate production and upregulates PFKFB3 expression in type 1 diabetic mice and aged mice

We next assessed lactate production in diabetic or aged mouse models. Consistent with our cell-based observations, GCGR mAb treatment significantly increased lactate levels in testicular tissues of diabetic mice (0.19 ± 0.036 mmol/g protein vs. 0.12 ± 0.019 mmol/g protein, P = 0.0038; Fig. 8A) and in aged mice (0.28 ± 0.13 mmol/g protein vs. 0.10 ± 0.030 mmol/g protein, P = 0.016; Fig. 8G). LDH activity was unaffected in both experimental models (Fig. 8B, H). Further molecular analysis confirmed that GCGR mAb treatment significantly upregulated PFKFB3 expression at both mRNA and protein levels in testicular tissues from diabetic mice and aged mice (Fig. 8C–E, I–K). LDHA expression remained unchanged (Fig. 8D, F, J, L). Notably, immunofluorescence staining showed that PFKFB3 signals in mouse testicular tissue were mainly localized to SOX9-positive Sertoli cells within seminiferous tubules (Fig. S10), supporting Sertoli cells as a key cellular compartment in which PFKFB3 may regulate glucagon-induced testicular glycolytic metabolism. These results suggest that GCGR signaling influences lactate production primarily through modulation of PFKFB3 expression in Sertoli cells.

Fig. 8.

GCGR blockade increases lactate production and upregulates PFKFB3 expression in the testes of diabetic mice and aged mice. Streptozotocin-induced diabetic mice (A–F) and 18-month-old male C57BL/6J (G–L) received treatment with a GCGR monoclonal antibody (mAb), or control (Ctrl). After a 4-week treatment, testes were harvested. Lactate content (A, G) and LDH activity (B, H) were measured using commercial colorimetric assay kits. The mRNA level of Pfkfb3 was assessed by reverse transcription qPCR (C, I). Protein levels of PFKFB3 and LDHA were assessed by western blot and quantified using Image J software (D–F, J–L). n = 3–5. Data were presented as mean ± SD. Statistical analyses were performed using two-tailed unpaired Student’s t-test or Welch’s t-test. *P < 0.05 and **P < 0.01 vs. the control group

PFKFB3 mediates glucagon effects on lactate production and BTB–associated gene expression in Sertoli cells

To confirm the functional role of PFKFB3 in glucagon-mediated changes, we established Pfkfb3 overexpression and knockdown models in Sertoli cells. Consistent with our data above, glucagon reduced lactate production, lowered PFKFB3 level, and downregulated the expression of the Sertoli-cell maturation marker Gata4 and BTB genes (Gja1, Cldn11) in Sertoli cells (Fig. 5O–T and Fig. 7). Pfkfb3 overexpression increased lactate production, and upregulated Gata4, Gja1 and Cldn11 mRNA expression, and largely rescued the suppressive effects of glucagon on these parameters (Fig. 9A–E).

Fig. 9.

PFKFB3 mediates the glucagon effects on lactate production and BTB–associated gene expression in Sertoli cells. Mouse Sertoli cell line TM4 cells were transfected with a Pfkfb3 overexpression plasmid (OE-Pfkfb3) or negative control (OE-NC), or with Pfkfb3 siRNA (si-Pfkfb3) or negative control siRNA (si-NC), and then treated with glucagon (GCG, 100 nmol/L) or vehicle control (Ctrl) for 24 h. A, F: PFKFB3 protein levels were measured by western blot and quantified by Image J software. B, G: Lactate content was measured using commercial colorimetric assay kits. C–E, H–J: The mRNA levels of the Sertoli cell specific marker Gata4 (C, H) and blood–testis barrier-related genes Gja1 (D, I) and Cldn11 (E, J) were measured by reverse transcription qPCR. n = 3–4. Data were presented as mean ± SD. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test or Welch’s ANOVA followed by Tamhane's T2 post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001

By contrast, Pfkfb3 knockdown markedly decreased the lactate production and reduced the expression levels of Gata4, Gja1 and Cldn11 compared to the control (Fig. 9F–J), with the similar effects to glucagon. Importantly, glucagon treatment in the Pfkfb3 knockdown group did not further amplify these effects (Fig. 9F–J), indicating that the suppressive impact of glucagon on lactate metabolism and BTB-related gene expression occurs predominantly through PFKFB3.

Collectively, these findings identify PFKFB3 as an essential mediator linking glucagon signaling to Sertoli cell metabolism and BTB integrity.

Pfkfb3 heterozygous deletion impairs sperm quality, reduces lactate production and BTB-associated markers in mice

To further validate the role of PFKFB3 in vivo, we analyzed reproductive phenotypes in Pfkfb3^+/− mice. Compared with wild-type littermates, Pfkfb3^+/− mice showed no significant change in body weight (Fig. 10A) but exhibited a moderate reduction in testicular weight (0.190 ± 0.012 g vs. 0.210 ± 0.016 g, P = 0.029, Fig. 10B).

Fig. 10.

Pfkfb3 heterozygous deletion impairs mice sperm quality. Eight-week-old male wild-type (WT) and Pfkfb3 heterozygous knockout (Pfkfb3^+/−) mice were used to assess sperm parameters (A–L) and testicular histology (M). A: Body weight. B: Testicular weight. C–L: Sperm concentration and motility were evaluated using a computer-assisted sperm analysis system. n = 5–7. C: Sperm concentration. D: Rapid progressive motility (grade A sperm). E: Progressive motility (grade A + B sperm). F: Total motility (grade A+B+C sperm). G: Velocity along the straight‑line path (VSL). H: Velocity along the curvilinear path (VCL). I: Velocity along the average path (VAP). J: Straightness (STR). K: Amplitude of lateral head displacement (ALH). L: Beat-cross frequency (BCF). M: Representative hematoxylin and eosin (HE) staining images of testicular sections. Scale bar = 50 μm. Data are presented as the mean ± SD. Statistical significance was assessed using two-tailed unpaired Student’s t-test or Welch’s t-test. *P < 0.05, **P < 0.01 vs. WT mice

CASA analysis showed impaired sperm quality in Pfkfb3^+/− mice. Pfkfb3^+/− mice exhibited a decreasing trend in sperm concentration and rapid progressive motility, while progressive motility and total motility (70.64 ± 6.14% vs. 81.76 ± 4.61%, P = 0.007) were significantly reduced (Fig. 10C–F). Among sperm kinetic parameters, VSL showed a decreasing trend, STR (0.430 ± 0.053% vs. 0.544 ± 0.036%, P = 0.002) and BCF (8.38 ± 0.93 HZ vs. 10.45 ± 0.73 HZ, P = 0.002) were significantly decreased in Pfkfb3^+/− mice (Fig. 10G–L). These findings indicate that partial loss of Pfkfb3 reduces sperm concentration and motility. HE staining did not reveal overt disruption of seminiferous tubule architecture in Pfkfb3^+/− mice compared with wild-type mice (Fig. 10M).

As expected, testicular lactate content was significantly decreased in Pfkfb3^+/− mice (0.226 ± 0.025 mmol/g protein vs. 0.305 ± 0.049 mmol/g protein, P = 0.013), while LDH activity remained unchanged (Fig. 11A, B). qPCR analysis showed reduced expression of Pfkfb3, the Sertoli-cell markers Wt1 and Gata4 (Fig. 11C–E), and the BTB-associated gene Tjp1 (Fig. 11F–I). At the protein level, western blot and immunofluorescence analyses consistently showed the reduced PFKFB3 expression and diminished ZO1 and Connexin 43 abundance in Pfkfb3^+/− testes (Fig. 11J–O). These results indicate that Pfkfb3 heterozygous deletion in vivo damages sperm quality, reduces lactate production and impairs Sertoli cells, just similar to the in vitro results, suggesting that targeting PFKFB3 and Sertoli cells might be a potential therapy for male infertility.

Fig. 11.

Pfkfb3 heterozygous deletion reduces lactate production and decreased BTB-associated genes in mice. Eight-week-old male wild-type (WT) and Pfkfb3 heterozygous knockout (Pfkfb3^+/−) mice were used to assess lactate metabolism (A–C), and gene expression (D–O). A–B: Testicular lactate content (A) and LDH activity (B) were measured using commercial colorimetric assay kits. n = 5. C–I: The mRNA levels of Pfkfb3 (C), the Sertoli-cell markers Wt1 and Gata4 (D, E), and BTB-associated genes Gja1, Cdh2, Cldn11 and Tjp1 (F–I) were analyzed by reverse transcription qPCR. n = 5. J–O: The protein levels of PFKFB3 (K), ZO1 (L) and Connexin 43 (M) in testes were measured by western blots and quantified by Image J software, n = 4. N–O: Representative immunofluorescence images showing ZO1 (N) and Connexin 43 (O) in testicular sections. Scale bar = 50 μm. Data are presented as the mean ± SD. Statistical significance was assessed using two-tailed unpaired Student’s t-test or Welch’s t-test. *P < 0.05, **P < 0.01 vs. WT mice

Discussion

Elevated glucagon signaling leads to diabetic complications [34, 35], and GCGR antagonism is a promising glucose-lowering strategy [13, 15, 29], but its reproductive effects in male have been unclear. In this study, we found that GCGR blockade increased sperm concentration and enhanced sperm motility in both metabolism stress (type 1 diabetic mice and naturally aged mice) and physiological (healthy young mice) conditions. GCGR inhibition also preserved seminiferous-tubule architecture and reduced oxidative stress and apoptosis in diabetic mice, while in aged mice it improved late spermatogenic features, as evidenced by a more organized seminiferous epithelium and more prominent PAS-positive acrosomal structures in developing spermatids. Although glucagon did not affect the cell proliferation of testicular cells (including spermatogonia cells, spermatocyte cells, Leydig cells, and Sertoli cells), it damaged the function of Sertoli cells, downregulated the expression of maturation markers and BTB-related genes. Mechanistically, glucagon suppressed PFKFB3 and lowered lactate production in Sertoli cells. These effects were largely reversed by GCGR mAb while partially attenuated by the GLP-1R antagonist, suggestive of the importance of GCGR for testicular protection. By using over-expression and knockdown of Pfkfb3 in glucagon-treated Sertoli cells and constructing Pfkfb3 heterozygous knockout mice, we confirmed the importance of PFKFB3 in lactate production, Sertoli-cell function and finally the sperm concentration and motility. Together, these findings implicate excessive GCGR signaling as a contributor to impaired spermatogenesis and suggest GCGR blockade and/or PFKFB3 activation as a strategy to restore Sertoli-cell support of germ cells and improve male reproductive function.

Antidiabetic treatments primarily target glycemic control, while insulin is recognized for its reproductive benefits, evidence for other drug classes is often limited or conflicting [36–39]. It is necessary to evaluate the effects of novel antidiabetic treatments, such as GCGR antagonists, on male reproduction. In diabetes (both type 1 and type 2 diabetes), excessive glucagon secretion and aberrant GCGR activation are major drivers of hyperglycemia [40]. GCGR blockade was established as a promising therapeutic strategy for achieving glycemic control, as demonstrated in both preclinical models and clinical studies [13, 19, 41, 42]. Given the broad tissue distribution of GCGR [8], its blockade may exert systemic effects beyond glucose regulation. GCGR antagonism was shown to exert some beneficial effects. For example, GCGR mAb treatment could increase pancreatic β-cell regeneration and survival [15, 29], improve cardiac function [18] and protect cardiac microvascular endothelial cells [43] in diabetic mouse models. However, GCGR’s role is not uniformly positive across all tissues. Kidney-specific deletion of GCGR leads to renal injury, indicating that complete GCGR blockade might carry organ-specific effects [44]. Given the links between metabolism and fertility, we hypothesized that chronic hyperglucagonemia or GCGR blockade might influence testicular function and the male reproductive system. In line with this hypothesis, our study demonstrates that GCGR mAb treatment exerts protective effects on male reproductive function in both diabetic mice and aged mice. GCGR mAb administration improved sperm motility in both models and increased sperm concentration while alleviating oxidative stress and apoptosis in the testes of diabetic mice. Surprisingly, GCGR mAb also improved sperm quality in healthy young mice, which suggests that glucagon/GCGR signaling may participate in male reproductive regulation beyond overt metabolic disease.

One thing needs to note is whether the benefits of systemic GCGR mAb treatment are mediated by the direct testicular signaling or through the improved systemic metabolism. There is no doubt that systemic GCGR blockade reduces hepatic glucose output and lowers glycemia, which could indirectly improve the testicular microenvironment by reducing hyperglycemia-driven oxidative stress, inhibiting inflammation, suppressing advanced glycation end-product formation, and maintaining BTB integrity and function [5]. To clarify the direct effect of glucagon signaling, at first, we confirmed the expression of GCGR in human testes and several testicular cells, including spermatogonia, spermatocytes, Leydig and Sertoli cells. Our in vitro assays showed that glucagon exposure did not directly affect sperm motility or cell proliferation of testicular cells. A clinical report indicated that glucagon did not alter reproductive hormone levels in healthy young men [45]. Similarly, we also found GCGR blockage did not change the sex hormone in diabetic mice and in aged mice. These findings suggest that glucagon is unlikely to impair male reproductive function through direct effects on sperm motility, testicular cell proliferation, or the systemic reproductive hormone axis.

Glucagon functions as a nutrient-sensitive endocrine signal. Hypoglycemia, prolonged fasting, exercise, and protein-rich meals stimulate glucagon secretion, whereas hyperglycemia and paracrine insulin/GLP-1 signaling suppress it [8]. Given the high metabolic demand of the testis, spermatogenesis relies on a stable supply of Sertoli-cell-derived nutrients, particularly lactate, to support developing germ cells [46, 47]. Thus, glucagon may act as a systemic nutrient-availability cue linking whole-body metabolic stress to testicular metabolic adaptation. Sertoli cells provide structural and metabolic support for germ cells, maintain the BTB, and are particularly vulnerable to metabolic stress [5, 48, 49]. We shifted our focus to investigate specific functional alterations in Sertoli cells.

Diabetes and aging were associated with reduced Sertoli cell glycolysis and lactate output, due to hyperglycemia-induced oxidative damage and impaired glucose utilization [33, 50, 51]. This compromised lactate supply was directly associated with impaired spermatogenesis, germ cell apoptosis, and reduced fertility [47, 52, 53]. In our study, we found that exposing Sertoli cells to glucagon significantly reduced lactate production (the primary energy substrate for germ cells, and has a known anti-apoptotic, germ cell-supportive effect [54]) and downregulated the key Sertoli cell transcripts—including the identity marker Gata4 and essential BTB junction genes (Cldn11 and Gja1), the products of which are essential for BTB integrity and immune privilege [33, 55]. Whereas these effects were largely reversed by GCGR mAb. Consistent with the in vitro findings, the GCGR mAb treated diabetic or aged mice showed elevated testicular lactate levels and restored expression of Sertoli cell genes, including Gata4 and Wt1, which are core Sertoli cell transcription factors that sustain cell maturation and junctional programs [56, 57], and BTB associated genes—Cldn11, Gja1, and Tjp1. Treated mice also exhibited improved seminiferous tubule integrity, reflecting a preservation of the structural support for spermatogenesis. Together, these results demonstrate that aberrant GCGR signaling can directly compromise Sertoli cell metabolic support and barrier function, pharmacological GCGR blockade recovers the Sertoli cell support, thereby safeguarding spermatogenesis and highlighting GCGR as a promising therapeutic target for metabolic male infertility.

Several antidiabetic drugs also affect the glycolytic flux and lactate production of Sertoli cells. Insulin was proved to be fundamental to this process, as its deficiency in type 1 diabetes reduces testicular lactate [58]. Insulin/insulin-like growth factor-1 signaling is also crucial for Sertoli cell proliferation. Genetic ablation of its receptors in Sertoli cells causes severely reduced cell numbers and testis size [59]. Metformin and thiazolidinediones (peroxisome proliferator-activated receptor γ agonists) were shown to enhance glycolysis and lactate production in cultured Sertoli cells [60, 61], increasing lactate availability for germ cells. Exenatide, a GLP-1R activator, upregulated glucose uptake and lactate secretion [62], while liraglutide, another GLP-1R activator, did not significantly change glucose consumption or lactate production in Sertoli cells, but it increased intracellular triacylglycerol levels and lipid droplet accumulation [63]. A sodium-glucose cotransporters 2 inhibitor dapagliflozin shifted Sertoli-cell metabolism from glycolysis towards fatty-acid β-oxidation and ketogenesis, despite upregulating glycolytic enzymes LDHA and the lactate transporter monocarboxylate transporter 4, resulting in reduced net lactate secretion [62]. Together, these observations indicate that antidiabetic and metabolic drugs differentially modulate Sertoli cell metabolism, underscoring the importance of considering their potential effects on germ cell metabolic support and reproductive function when such agents are used for disease treatment.

Notably, antidiabetic therapies were shown to exert context-dependent effects on the reproductive function. Insulin consistently demonstrating protective effects on male reproductive function both in physiological and pathological conditions [33]. Insulin signaling is essential for Sertoli cell functions, including androgen-binding protein production, lactate secretion, and the synthesis of structural and functional proteins as well as DNA. Insulin also supports Leydig cell growth and testosterone synthesis, thereby facilitating normal spermatogenesis [64]. Additionally, insulin enhances sperm motility, the acrosome reaction, and fertilization capability [36]. However, evidence for other drugs is inconsistent. Under diabetic conditions, metformin intervention has been reported to improve testicular function, sperm quality, and spermatogenesis [39, 65]. In contrast, under normal (non-diabetic) conditions, metformin reduced cell number and suppressed proliferation of Sertoli cells [66, 67]. Similarly, animal studies have reported that sodium-glucose cotransporter 2 inhibitors can reduce diabetes-associated oxidative stress in testes, improve testicular dysfunction and sperm quality [68], whereas short-term sodium-glucose cotransporter 2 blockade in men without diabetes did not change serum level of inhibin B, which is the Sertoli-derived marker of spermatogenic activity and FSH feedback, suggesting no acute secretory impairment [69]. Thiazolidinediones could protect the BTB from metabolic stress [7, 70], but excessive peroxisome proliferator-activated receptor γ activation in the testis microenvironment could induce adipogenic gene programs, raising lipid accumulation in Sertoli cells and causing damage to proliferation [71, 72]. Consistent with this context-dependent effect, although GCGR mAb treatment exhibited protective effects on Sertoli cells and increased sperm motility in aged mice, no significant improvement in semen concentration and testicular structure was observed, possibly because metabolic impairment in this aging model was relatively mild. Previous studies using mice with systemic Gcgr deficiency reported that males remained fertile under natural mating conditions [19]. Our study further evaluated the spermatogenesis, semen parameters and testicular histology and found the constitutive Gcgr knockout male displayed lower sperm concentration and motility althouth the deficiency did not affect seminiferous-tubule architecture and the live birth outcome. These results suggest GCGR is necessary for spermatogenesis during embryonic develoment. The effects of GCGR signaling suppression on male reproduction depend on when and how GCGR signaling is inhibited, as pharmacological blockade initiated in adulthood may not fully reflect the effects of constitutive systemic Gcgr deletion established from embryonic development. Collectively, these findings suggest that the effects of antidiabetic interventions on male reproduction are highly context dependent, with protective effects most evident under diabetic or metabolically stressed conditions. In contrast, their effects under euglycemic conditions or constitutive genetic pathway disruption may differ substantially, highlighting the need to consider metabolic status, drug class, and timing of intervention when evaluating reproductive outcomes.

To investigate the possible mechanism of glucagon signaling on Sertoli cells, we profiled the expression of transporters, key glycolytic genes and lactate metabolism-related genes, and identified the glycolytic regulator PFKFB3, the predominant isoenzyme controlling glycolytic flux in Sertoli cells and in mouse testes [73]. By using overexpression and knockdown, we confirmed PFKFB3 as a critical node in regulating lactate production and cell function induced by glucagon in Sertoli cells. Besides, Pfkfb3 heterozygous deletion reduced testicular lactate production, downregulated Sertoli-cell markers and BTB-associated genes, decreased testicular weight, and inhibited sperm motility in vivo. These evidence supports a Sertoli-cell-related GCGR–PFKFB3–lactate axis for testicular protection. PFKFB3 is also associated with glycolytic remodeling in multiple contexts. In macrophages, PFKFB3 and glycolysis is concomitant with the M2-to-M1 phenotype conversion, and interventions (e.g. sodium-glucose cotransporters 2 inhibitors) targeting PFKFB3 to regulate the glycolytic flux could change the macrophage functional state [74]. In kidney proximal tubular cells, PFKFB3 induction is accompanied by increased lactate accumulation and protein lactylation, leading to NF-κB activation and the consequent pro-inflammatory and pro-fibrotic responses [75]. These findings support that PFKFB3 functions not only as a glycolytic regulator but also as a metabolic node linking glycolytic remodeling to cellular functional changes. In addition to PFKFB3, other glycolytic regulators can also function within Sertoli cells to support spermatogenesis. For instance, phosphoglycerate mutase 1, a key glycolytic enzyme, regulates energy metabolism in Sertoli cells, thereby influencing their capacity to support spermatogonia stem cell proliferation and differentiation [76]. All these findings suggest that targeting glycolysis (including PFKFB3-driven glycolysis) might represent a potential intervention to enhance Sertoli-cell function and, consequently, support sperm quality and overall testicular function.

In Sertoli cells, PFKFB3 has also been shown to be responsive to hormonal regulation. FSH, an essential hormone for Sertoli-cell proliferation, maturation, survival, and early spermatogenesis [77–79], upregulates Pfkfb3 transcription and activity, and stimulates a high glycolytic rate and lactate production. PFKFB3 inhibition abolishes the FSH-induced lactate increment in Sertoli cells [80]. In this study, glucagon was identified as an additional upstream regulator hormone of PFKFB3 in Sertoli cells, providing a potential link between glucagon signaling, Sertoli-cell lactate production, and BTB-related gene expression. What is the possible pathway between glucagon and PFKFB3? Glucagon can activate both GCGR and GLP-1R under certain conditions [29, 81]. We detected GCGR and GLP-1R were expressed in Sertoli cells. GCGR mAb totally reversed while GLP-1R antagonist partially attenuated the glucagon-induced changes in intracellular cAMP, lactate production, Pfkfb3 expression, and Sertoli-cell and BTB-associated markers. These results suggest that GLP-1R-related signaling does not fully explain the PFKFB3–lactate–BTB phenotype while GCGR accounts for most of the effects. Besides, in our previous study, pharmacological activation of GLP-1R with liraglutide improved glycemic parameters in diabetic and aged mice, but did not improve sperm concentration or motility parameters [82]. These in vivo data further support the view that GLP-1R activation alone is insufficient to rescue sperm quality. However, the downstream mechanisms linking GCGR activation to PFKFB3 suppression remain unclear. Future studies using Sertoli-cell-specific GCGR/GLP-1R loss-of-function models, PKA/EPAC/CREB pathway perturbation, Pfkfb3 promoter-reporter assays, and direct PFKFB3 activity assays will be required to define the mechanistic signaling pathway.

Several limitations should be acknowledged. First, GCGR mAb was administered for only four weeks in this study. Therefore, the long-term durability of its reproductive benefits and potential endocrine adaptations, such as hyperglucagonemia, altered amino acid metabolism, and pancreatic α-cell remodeling, need further evaluation. Besides, although the main phenotypic outcomes were assessed in all animals, some histological, biochemical, and molecular analyses were conducted with smaller sample subsets due to limited testicular tissue availability. Future studies with larger cohorts will be concluded to validate these findings. Second, we assessed sperm parameters, testicular histology, apoptosis, oxidative stress, lactate production, and Sertoli-cell/BTB-related markers as reproductive indicators. Direct fertility outcomes, functional BTB assays, and real-time glycolytic flux measurements, which are important for confirming the functional relevance of the GCGR–PFKFB3–lactate axis, were not performed. Third, the current in vivo data cannot fully distinguish testis-intrinsic GCGR signaling from indirect benefits secondary to metabolic improvement. A glucose-lowering comparator might be helpful to answer this question. However, it is still imperfect, as many antidiabetic therapies can independently affect male reproduction or Sertoli-cell metabolism. Future studies using glycemia-matched designs together with adult Sertoli-cell-specific Gcgr or Pfkfb3 manipulation will help resolve this issue. Fourth, the STZ-induced diabetic model may involve direct STZ-related reproductive toxicity independent of hyperglycemia. Nevertheless, our findings are supported by additional non-STZ settings, including naturally aged mice, healthy young mice, and STZ-free TM4 Sertoli-cells. Finally, the human relevance of this pathway remains preliminary. Owing to ethical and practical constraints, interventional studies involving glucagon or GCGR mAb treatment in humans, followed by testicular tissue collection, were not feasible. However, our human testicular localization data provide initial anatomical evidence for the relevance of glucagon/GCGR signaling in human testes. Future functional validation using primary human Sertoli cells, human testicular organoid, and clinical semen cohorts will determine whether the GCGR–PFKFB3–lactate pathway is conserved in humans.

Conclusions

Our study identified excessive glucagon–GCGR signaling as a previously underappreciated contributor to metabolic impairment of Sertoli cells and defective spermatogenesis. By linking GCGR activity to PFKFB3-dependent glycolytic flux and lactate production, we uncovered a metabolic mechanism through which GCGR mAb preserves Sertoli-cell support, BTB integrity, and male reproductive function under diabetic and aging conditions. These findings not only expand the biological scope of GCGR signaling beyond systemic glucose regulation but also highlight Sertoli-cell glycolysis as a potential therapeutic target for treating metabolism-associated male infertility.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

ALH

Amplitude of lateral head displacement

BAX

Bcl-2–associated X protein

BCF

Beat-cross frequency

BCL2

B-cell lymphoma 2

BTB

Blood–testis barrier

CASA

Computer-assisted sperm analysis

CCK-8

Cell counting kit-8

Cdh2

Cadherin 2

Cldn11

Claudin 11

DAPI

4’,6-diamidino-2-phenylindole

ELISA

Enzyme-linked immunosorbent assay

FSH

Follicle-stimulating hormone

GCGR

Glucagon receptor

Gja1

Gap junction protein alpha 1

GLP-1R

Glucagon-like peptide-1 receptor

HE

Hematoxylin and Eosin

Hk2

Hexokinase 2

LDH

Lactate dehydrogenase

LH

Luteinizing hormone

mAb

monoclonal antibody

MDA

Malondialdehyde

PFKFB3

6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3

Pkm2

Pyruvate kinase isozyme M2

qPCR

quantitative polymerase chain reaction

SD

Standard deviation

SOD

Superoxide dismutase

STR

Straightness

Tjp1

Tight junction protein 1

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick-end labeling

VAP

Velocity along the average path

VCL

Velocity along the curvilinear path

VSL

Velocity along the straight-line path

Author contributions

L.X., F.L. and D.Y. contributed equally to this work. L.X. performed the majority of the experiments, F.L. and D.Y. performed the cell-based experiments, L.X. and F.L. collected and analyzed the data, conducted literature review, drafted the manuscript, revised the manuscript, and coordinated data verification and integrity checks. D.Y. and T.W. contributed to animal experiments and assisted with data analysis. C.S. participated in cell-based experiments and data analysis. Z.Z. contributed to data verification and manuscript proofreading. R.W. conceived and designed the study, supervised the overall research progress and critically revised the manuscript. All authors have read and approved the final manuscript.

Funding

This work was supported by the Noncommunicable Chronic Diseases-National Science and Technology Major Project (2023ZD0507000), the National Key Research & Developmental Program of China (2022YFC2702600), the special fund of the National Clinical Key Specialty Construction Program, P. R. China (2023), and the Talent Project of Clinical Key Project of Peking University Third Hospital.

Data availability

The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The tissue sections were provided by Prof. Hui Jiang. The study was approved by the Peking University Third Hospital Medical Science Research Ethics Committee (approval No. IRB00006761-M2022692). All animal experiments were conducted at Peking University Health Science Center and approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center (Approval number PUIRB-LA2023522). All procedures involving animals were performed in accordance with the Chinese national standard “Laboratory animal—Guideline for ethical review of animal welfare” (GB/T 35892 − 2018), the institutional guidelines for the care and use of laboratory animals, and the internationally recognized 3R principles (Replacement, Reduction, Refinement).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.